The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.

Serotonin 5-HT 2B receptors are often coexpressed with 5-HT 1B receptors, and cross-talk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT 1B and 5-HT 2B receptor cross-talk have not been elucidated. We hypothesized that 5-HT 2B and 5-HT 1B receptors each affect the others' signaling by modulating the others' trafficking. We thus examined the agonist stimulated internalization kinetics of fluorescent protein-tagged 5-HT 2B and 5-HT 1B receptors when expressed alone and upon coexpression in LMTK Ϫ murine fibroblasts. Time-lapse confocal microscopy and wholecell radioligand binding analyses revealed that, when expressed alone, 5-HT 2B and 5-HT 1B receptors displayed distinct half-lives. Upon coexpression, serotonin-induced internalization of 5-HT 2B receptors was accelerated 5-fold and was insensitive to a 5-HT 2B receptor antagonist. In this context, 5-HT 2B receptors did internalize in response to a 5-HT 1B receptor agonist. In contrast, coexpression did not render 5-HT 1B receptor internalization sensitive to a 5-HT 2B receptor agonist. The altered internalization kinetics of both receptors upon coexpression was probably not due to direct interaction because only low levels of colocalization were observed. Antibody knockdown experiments revealed that internalization of 5-HT 1B receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, whereas that of 5-HT 2B receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon coexpression, serotonin-induced 5-HT 2B receptor internalization became partially Caveolin1-dependent, and serotonin-induced 5-HT 1B receptor internalization became entirely Caveolin1-independent in a protein kinase Cdependent fashion. In conclusion, these data demonstrate that coexpression of 5-HT 1B and 5-HT 2B receptors influences the internalization pathways and kinetics of both receptors.Serotonin (5-hydroxytryptamine, 5-HT) is a potent vasoactive molecule and also a major neurotransmitter in both the central and peripheral nervous systems (Hoyer et al., 2002). All 5-HT receptors, except 5-HT 3 , belong to the rhodopsinlike G protein-coupled receptor (GPCR) superfamily. The 5-HT 1B , 5-HT 2B , and 5-HT 2A receptors were found in endothelial and smooth muscle cells from several human and mouse arteries at mRNA, protein and functional levels (Ullmer et al., 1995;Watts et al., 1996). In addition, varioushuman meningeal tissues have been found to coexpress 5-HT 1B and 5-HT 2B receptor mRNA (Schmuck et al., 1996). Thus, given their coexpression and their role in regulating smooth muscle contractility (Banes and Watts, 2003), 5-HT 1B and 5-HT 2B receptors have been implicated in the pathogenThis work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Pierre et Marie Curie, the Université Louis Pasteur, and by grants from the Fondation de France, the Fondation pour la ...

(i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.

Although potentially implicated in several physiological functions, few functional mutations have been identified in the human 5-hydroxytryptamine (HT) 2B receptor gene. A heterozygous mutation R393X in the 5-HT 2B receptor was recently identified in a patient diagnosed with pulmonary hypertension after intake of the anorexigenic dexfenfluramine. Although reported to generate a lack of function, this C terminus-truncated 5-HT 2B receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative modifications in transduction of the R393X-mutated 5-HT 2B receptor. In stably transfected cells, we confirmed the loss of inositol 1,4,5-trisphosphate stimulation caused by the G ␣q uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of nitric-oxide synthase stimulation. However, the truncated R393X receptor presented 1) a strong gain of efficacy in cell proliferation as assessed by mitogen-activated protein kinase activity and thymidine incorporation, 2) a preferential coupling to G ␣13 as shown by blocking antiserum, and 3) an apparent lack of internalization upon agonist stimulation as observed by confocal microscopy. This work demonstrates that, in the 5-HT 2B receptor, the C terminus, including the palmitoylation and phosphorylation sites, is absolutely required for proper transduction and internalization. For the first time, we show that the lack of C terminus can generate a switch of coupling to G ␣13 , a reduced NO synthase activation, and an increase in cell proliferation. All these modifications are relevant in pathophysiological vasoconstriction.Knowledge of 5-HT 2B receptor genetic variants is of the utmost interest in view of the involvement of these receptors in various pathologies (Roth and Shapiro, 2001). Few studies have investigated mutations in the 5-HT 2B receptor gene (MIM 601122). It was identified as a candidate gene in obsessive-compulsive disorder: one single nucleotide polymorphism was described in intron 1, but no evidence for functional mutation was found in the coding regions of 5-HT 2B receptor (Kim et al., 2000). More recently, by investigating the 5-HT 2B receptor gene in patients who developed pulmonary hypertension after intake of fenfluramines, a heterozygous mutation was found in one female who 5 years before followed a 9-month anorexigenic treatment (Blanpain et al., 2003). The reported mutation consists of a C/T transition This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Hôpital Universitaire de Strasbourg, the Université Louis Pasteur, and by grants from the Fondation de France, the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer, the French ministry of research (Actions Concertées Incitatives), and the European Community.